1. Field of the Invention
The present invention discloses a new series of derivatives of adenine and adenosine, which inhibit adenylyl cyclase activity. These newly synthesized compounds include the most potent in vitro inhibitors of adenylyl cyclases known. The present invention also discloses a method for measuring the inhibition of adenylyl cyclase activity.
2. Description of the Related Art
Adenosine-3':5'-cyclic monophosphate (cAMP) is the second messenger involved in signal transduction for numerous neurotransmitters and hormones.sup.1. The cAMP pathway can be regulated pharmacologically by many drugs that are of particular value in the treatment of many diseases, and therefore there is much current interest in identifying new agents acting on this pathway. Regulation of this pathway can be achieved through changes in the activities of cAMP-phosphodiesterases.sup.2, cAMP-dependent protein kinases.sup.3, or adenylyl cyclases.sup.1.
Adenylyl cyclases are a family of enzymes that:
a) catalyze the formation of cAMP from adenosine-5'-triphosphate (5'ATP); PA1 b) mediate the physiological effects of numerous hormones and neurotransmitters; and PA1 c) belong to a super family of membrane-bound transporters and channel proteins. PA1 a) inhibition is non-competitive with respect to substrate.sup.9-16, consistent with and corroborated by a number of other studies indicating that inhibition is at a site distinct from catalysis.sup.17 ; PA1 b) potencies of "P"-site inhibitory ligands are substantially greater with stimulated forms of adenylyl cyclase than with the basal enzyme.sup.12-14,16-21 ; PA1 c) potency of inhibition is isozyme-dependent.sup.21,22, suggesting the possibility that the physiological importance of this inhibitory mechanism is dictated by the isozyme expressed in a given tissue; PA1 d) inhibition has a striking requirement for an intact adenine moiety in that inosine, guanosine, cytosine, thymidine, and uridine are inactive, and for adenosine N.sup.6 - and C(8)-substitutions are not tolerated, N(3)-, N(7)-, and N(9)-deaza analogues are inactive.sup.19, though 2-fluoroadenosine and 2-fluoro-2'-deoxyadenosine are inhibitory.sup.23 ; PA1 e) inhibition exhibits a strong preference for 3'-phosphate, but not cyclic phosphates (3':5' or 2':3').sup.11,19 ; PA1 f) 2'-deoxy- and especially 2',5'-dideoxy-ribosyl compounds.sup.9,10,13,19,22,24 exhibit enhanced inhibitory potency; and PA1 g) there is tolerance for significant modifications of the ribose moiety, especially tolerance for large substitutions at the 3'-position, as evidenced particularly by the inhibitory effectiveness of 2'-deoxy-adenosine-di-nucleotides and 2'-deoxyadenosine-penta-nucleotides, 3'-(4-fluorosulfonylbenzoyl)-2',5'-dideoxyadenosine (2',5'-dd-3'FSB-Ado).sup.17,19, and 3'-succinyl-2',5'-dideoxyadenosine.sup.19.
Numerous drugs have been developed as therapeutic agents that inhibit cyclic nucleotide phosphodiesterases.sup.4, the effects of which are to raise cellular cAMP levels in tissues and organs on which they act. For example, theophylline, an inhibitor of all isozyme families of phosphodiesterases, is used clinically to treat asthma.sup.5. Rolipram, an inhibitor of type IV phosphodiesterase, is used in the treatment of depression.sup.6. And several inhibitors of type III phosphodiesterase have been used clinically to treat patients with moderate to severe heart failure. These latter drugs enhance cardiac index without elevating mean arterial blood pressure and lowering systemic vascular resistance.sup.4 and hence, have significant advantages over .beta.-agonists and digitalis.
However, drugs that act directly on adenylyl cyclases have been less well explored, although agents which indirectly activate or indirectly inhibit the enzyme are commonly used in the treatment of disease. For example, drugs of the class .beta.-blockers are commonly used to treat hypertension and these act to inhibit adenylyl cyclase indirectly by blocking the stimulatory effects of the sympathetic nervous system to activate adenylyl cyclase in the heart, thereby reducing cardiac output.sup.7. Thus, agents that reduce adenylyl cyclase activity directly can have a similar cardiac-sparing effect and reduced cardiomyopathy and heart failure. Adenylyl cyclases are potently and directly inhibited by analogues of adenosine, via a specific domain.sup.8. This binding domain is referred to as the "P"-site from an evident requirement for an intact purine moiety.sup.9. The essential features include the following:
Tolerated modifications to or replacements of the ribosyl moiety include: 9-.beta.-D-arabinofuranosyl-adenine (IC.sub.50 .about.100 .mu.M), 9-.beta.-D-xylofuranosyl-adenine (IC.sub.50 .about.25 .mu.M).sup.9 ; erit-adenine (IC.sub.50 .about.6-12 .mu.M).sup.24 ; 9-(tetrahydro-5-methyl-2-furyl)-adenine (IC.sub.50 .about.8 .mu.M), 9-(tetrahydro-2-furyl)-adenine (IC.sub.50 .about.10 .mu.M), 9-cyclopentyl-adenine (IC.sub.50 .about.20 .mu.M), and 9-furfuryl-adenine (IC.sub.50 .about.26 .mu.M).sup.10.
In this patent application, we describe the synthesis of a new series of analogues and derivatives of adenine and of adenosine, which interact with the "P"-site specific binding domain on adenylyl cyclases. In general, these newly synthesized compounds include the most potent inhibitors of adenylyl cyclases known and are more potent than hormones whose effects must be mediated by G-proteins.
The synthesis of 3'-nucleotides has received considerably less attention than has the synthesis of the corresponding 5'-polyphosphates since the former are more difficult to synthesize and as their biological role is not as well described.sup.25-33. The only similar nucleotides are the Magic Spots, which are naturally occurring guanosine derivatives polyphosphorylated in both 3'- and 5'-positions.sup.34-38.
The adenine derivatives of the present invention inhibit adenylyl cyclases directly. The cell permeable variants, e.g. 9-(cyclopentyl)-2-fluoroadenine, inhibit the enzyme in intact cells and tissues and one such P-site inhibitor, 2',5'-dideoxy-adenosine has been used to counteract the effects of cholera intoxication in intestinal epithelium.sup.39. In addition, the cell impermeable variants, e.g. the adenine nucleoside 3'-polyphosphates, inhibit adenylyl cyclase when microinjected into cells or when introduced into cells by the application of liposome technology.
The compounds of the present invention are also useful in the preparation of covalent affinity probes and affinity chromatography matrices. Moreover, members of this new class of drug are important to many aspects of biology, biochemistry, pharmacology, and therapeutics and will find use in the treatment of various diseases and, more specifically, treatment of cardiovascular diseases.